PROJECT SUMMARY/ABSTRACT Multiple Sclerosis (MS) is characterized by focal inflammatory infiltrates of the CNS, demyelinating lesions and axonal damage, leading to neurologic dysfunction. Cells of the innate and adaptive immune system are involved in MS pathogenesis. MS is also characterized by production of cytokines that activate immune cells, including IFN-, IL-6, GM-CSF, IL-12 and IL-23. The JAK/STAT pathway is used by cytokines for signaling, and is critical for the development, regulation and termination of immune responses. In the previous funding period, we developed a model of Experimental Autoimmune Encephalomyelitis (EAE) in which the JAK/STAT pathway is aberrantly activated in myeloid cells, with functional consequences for Th1 and Th17 cell differentiation. This model presents as a non-resolving form of atypical EAE. In this atypical model with heightened activation of the JAK/STAT pathway, we demonstrate that AZD1480, an inhibitor of the JAK/STAT pathway, suppresses disease severity, prevents entry of immune cells into the CNS, suppresses differentiation of Th1 and Th17 cells, decreases STAT activation, and reduces expression of cytokines/chemokines. Furthermore, we have tested AZD1480 in four other EAE models (Classical, Relapsing-Remitting (RR), Adoptive Transfer of Th1 or Th17 cells) and observed striking clinical efficacy. Another JAK/STAT inhibitor, AZD5776, also inhibits EAE clinical symptoms. We hypothesize that inappropriate activation of the JAK/STAT pathway promotes aberrant responses of cells of the innate and adaptive immune systems, leading to the neuroinflammation seen in MS, and that therapeutic intervention of this pathway will alter the progression of MS. In this revised, competing renewal, we will evaluate two distinct JAK inhibitors, AZD1480 and AZD5776, in five EAE models. AZD1480 is a JAK1 and JAK2 inhibitor, and AZD5776 inhibits JAK1. In Aim 1, the therapeutic efficacy of targeting the JAK/STAT pathway in active EAE (classical, atypical, RR) will be examined. We will evaluate 1) which JAKs/STATs are involved in disease pathogenesis; 2) in which cell type(s) (CD4+ T-cells, macrophages, microglia, DCs) activation of the JAK/STAT pathway is detrimental; and 3) the molecular basis for the therapeutic benefit of the JAK inhibitors. In Aim 2, the role of the JAK/STAT pathway in regulating the pathogenic potential of Th1 and Th17 cells will be tested. Both Th1 and Th17 cells have the capacity to induce CNS autoimmunity, but have differential pathogenic mechanisms. Our preliminary results indicate that AZD1480 is effective in inhibiting both Th1-induced and Th17-induced EAE. We will assess how in vivo treatment with AZD1480/AZD5776 in the context of MOG-immunization regulates the developmental program and/or pathogenicity of Th1 and Th17 cells. In Aim 3, the mechanism by which the JAK inhibitors AZD1480 and AZD5776 regulate immune cell function will be examined. Macrophages and DCs obtained from nave mice treated with JAK inhibitors will be evaluated for their antigen presentation capability, polarization into proinflammatory or anti-inflammatory phenotypes, and potential neuroprotective properties. CD4+ T-cells derived from JAK inhibitor treated mice will be examined for their subsequent ability to differentiate into pathogenic and/or protective T-cell subsets. These studies will establish the pre-clinical foundation necessary to ultimately test inhibitors of the JAK/STAT pathway in human MS.